Screening assays for modulators of human kinesin protein HsKif16b

ABSTRACT

The invention provides isolated nucleic acid and amino acid sequences of HsKif16b, antibodies to HsKif16b, methods of screening for HsKif16b modulators using biologically active HsKif16b, and kits for screening for HsKif16b modulators.

FIELD OF THE INVENTION

The invention provides isolated nucleic acid and amino acid sequences ofHsKif16b, methods of detecting HsKif16b and screening for HsKif16bmodulators using biologically active HsKif16b, and kits for screeningfor HsKif16b modulators.

BACKGROUND OF THE INVENTION

The kinesin superfamily is an extended family of related microtubulemotor proteins. It can be classified into at least 8 subfamilies basedon primary amino acid sequence, domain structure, velocity of movement,and cellular function. This family is exemplified by “true” kinesin,which was first isolated from the axoplasm of squid, where it isbelieved to play a role in anterograde axonal transport of vesicles andorganelles (see, e.g., Goldstein, Annu. Rev. Genet. 27:319-351 (1993)).Kinesin uses ATP to generate force and directional movement associatedwith microtubules. The nematode Unc-104 kinesin-like protein defines adistinct kinesin subfamily whose members may function monomerically.Members of this subfamily are important for synaptic transport andmitochondrial translocation. Nematodes with mutations in the Unc-104gene exhibit defects in locomotion and feeding behaviors and, at themolecular level, in synaptic vesicle transport.

The discovery of a new kinesin motor protein, and more particularly, onein the Unc-104 subfamily, and the polynucleotides encoding it satisfiesa need in the art by providing new compositions which are useful in thediagnosis, prevention, and treatment of cancer, neurological disorders,and disorders of vesicular transport.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of a new human kinesinmotor protein, HsKif16b (also known as J777L9), the polynucleotideencoding HsKif16b, and the use of these compositions for the diagnosis,treatment, or prevention of cancer, neurological disorders, anddisorders of vesicular transport.

In one aspect, the invention provides an isolated nucleic acid sequenceencoding a kinesin superfamily motor protein, wherein the motor proteinhas the following properties: (i) the protein's activity includesmicrotubule stimulated ATPase activity; and (ii) the protein has asequence that has greater than 70% amino acid sequence identity to SEQID NO:2 or SEQ ID NO:4 as measured using a sequence comparisonalgorithm. In one embodiment, the protein further specifically binds topolyclonal antibodies raised against SEQ ID NO:2 or SEQ ID NO:4.

In one embodiment, the nucleic acid encodes HsKif16b or a fragmentthereof. In another embodiment, the nucleic acid encodes SEQ ID NO:2 orSEQ ID NO:4. In another embodiment, the nucleic acid has a nucleotidesequence of SEQ ID NO:1 or SEQ ID NO:3.

In one aspect, the nucleic acid comprises a sequence which encodes anamino acid sequence which has greater than 70% sequence identity withSEQ ID NO:2 or SEQ ID NO:4, preferably greater than 80%, more preferablygreater than 90%, more preferably greater than 95% or, in anotherembodiment, has 98 to 100% sequence identity with SEQ ID NO:2 or SEQ IDNO:4.

In one embodiment, the nucleic acid comprises a sequence which hasgreater than 55 or 60% sequence identity with SEQ ID NO:1 or SEQ IDNO:3, preferably greater than 70%, more preferably greater than 80%,more preferably greater than 90 or 95% or, in another embodiment, has 98to 100% sequence identity with SEQ ID NO:1 or SEQ ID NO:3. In anotherembodiment provided herein, the nucleic acid hybridizes under stringentconditions to a nucleic acid having a sequence or complementary sequenceof SEQ ID NO:1 or SEQ ID NO:3.

In another aspect, the invention provides an expression vectorcomprising a nucleic acid encoding a kinesin superfamily motor protein,wherein the motor protein has the following properties: (i) theprotein's activity includes microtubule stimulated ATPase activity; and(ii) the protein has a sequence that has greater than 70% amino acidsequence identity to SEQ ID NO:2 or SEQ ID NO:4 as measured using asequence comparison algorithm. The invention further provides a hostcell transfected with the vector.

In another aspect, the invention provides an isolated kinesinsuperfamily motor protein, wherein the protein has one or more of theproperties described above. In one embodiment, the protein specificallybinds to polyclonal antibodies generated against a motor domain, taildomain or other fragment of HsKif16b. In another embodiment, the proteincomprises an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4.

In one aspect, the protein provided herein comprises an amino acidsequence which has greater than 70% sequence identity with SEQ ID NO:2or SEQ ID NO:4, preferably greater than 80%, more preferably greaterthan 90%, more preferably greater than 95% or, in another embodiment,has 98 to 100% sequence identity with SEQ ID NO:2 or SEQ ID NO:4.

The invention features a substantially purified polypeptide comprisingthe amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4 or a fragmentthereof and more particularly, the motor domain of the amino acidsequence of SEQ ID NO:2 or SEQ ID NO:4 or a fragment thereof.

In one embodiment, the present invention provides a method ofidentifying a candidate agent as a modulator of the activity of a targetprotein. The method comprises adding a candidate agent to a mixturecomprising a target protein which directly or indirectly produces ADP orphosphate, under conditions that normally allow the production of ADP orphosphate. The method further comprises subjecting the mixture to areaction that uses said ADP or phosphate as a substrate under conditionsthat normally allow the ADP or phosphate to be utilized and determiningthe level of activity of the reaction as a measure of the concentrationof ADP or phosphate. A change in the level between the presence andabsence of the candidate agent indicates a modulator of the targetprotein.

The phrase “use ADP or phosphate” means that the ADP or phosphate aredirectly acted upon by detection reagents. In one case, the ADP, forexample, can be hydrolyzed or can be phosphorylated. As another example,the phosphate can be added to another compound. As used herein, in eachof these cases, ADP or phosphate is acting as a substrate.

Preferably, the target protein either directly or indirectly producesADP or phosphate and comprises a motor domain. More preferably, thetarget protein comprises a kinesin superfamily motor protein asdescribed above and most preferably, the target protein comprisesHsKif16b or a fragment thereof.

Also provided are modulators of the target protein including agents forthe treatment of cellular proliferation, including cancer, hyperplasias,restenosis, cardiac hypertrophy, immune disorders and inflammation. Theagents and compositions provided herein can be used in variety ofapplications which include the formulation of sprays, powders, and othercompositions. Also provided herein are methods of treating cellularproliferation disorders such as cancer, hyperplasias, restenosis,cardiac hypertrophy, immune disorders and inflammation, for treatingdisorders associated with HsKif16b activity, and for inhibitingHsKif16b.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, and 1C show an embodiment of a nucleic acid sequenceencoding a particularly preferred fragment of HsKif16b (SEQ ID NO:1).

FIGS. 2A, 2B, and 2C show the predicted amino acid sequence of aparticularly preferred fragment of HsKif16b (SEQ ID NO:2).

FIG. 3 shows an embodiment of a nucleic acid sequence encoding aHsKif16b motor domain fragment (SEQ ID NO:3).

FIG. 4 shows the predicted amino acid sequence of HsKif16b motor domainfragment (SEQ ID NO:4).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

“ADP” refers to adenosine diphosphate and also includes ADP analogs,including, but not limited to, deoxyadenosine diphosphate (dADP) andadenosine analogs. “Antibody” refers to a polypeptide substantiallyencoded by an immunoglobulin gene or immunoglobulin genes, or fragmentsthereof which specifically bind and recognize an analyte (antigen). Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Light chains are classified aseither kappa or lambda. Heavy chains are classified as gamma, mu, alpha,delta, or epsilon, which in turn define the immunoglobulin classes, IgG,IgM, IgA, IgD and IgE, respectively. The term antibody also includesantibody fragments either produced by the modification of wholeantibodies or those synthesized de novo using recombinant DNAmethodologies.

An “anti-HsKif16b” antibody is an antibody or antibody fragment thatspecifically binds a polypeptide encoded by the HsKif16b gene, cDNA, ora subsequence thereof.

“Biologically active” target protein refers to a target protein that hasone or more of kinesin protein's biological activities, including, butnot limited to microtubule stimulated ATPase activity, as tested, e.g.,in an ATPase assay. Biological activity can also be demonstrated in amicrotubule gliding assay or a microtubule binding assay. “ATPaseactivity” refers to ability to hydrolyze ATP. Other activities includepolymerization/depolymerization (effects on microtubule dynamics),binding to other proteins of the spindle, binding to proteins involvedin cell-cycle control, or serving as a substrate to other enzymes, suchas kinases or proteases and specific kinesin cellular activities, suchas chromosome congregation, axonal transport, etc.

“Biological sample” as used herein is a sample of biological tissue orfluid that contains a target protein or a fragment thereof or nucleicacid encoding a target protein or a fragment thereof. Biological samplesmay also include sections of tissues such as frozen sections taken forhistological purposes. A biological sample comprises at least one cell,preferably plant or vertebrate. Embodiments include cells obtained froma eukaryotic organism, preferably eukaryotes such as fungi, plants,insects, protozoa, birds, fish, reptiles, and preferably a mammal suchas rat, mice, cow, dog, guinea pig, or rabbit, and most preferably aprimate such as chimpanzees or humans.

A “comparison window” includes reference to a segment of any one of thenumber of contiguous positions selected from the group consisting offrom 25 to 600, usually about 50 to about 200, more usually about 100 toabout 150 in which a sequence may be compared to a reference sequence ofthe same number of contiguous positions after the two sequences areoptimally aligned. Methods of alignment of sequences for comparison arewell-known in the art. Optimal alignment of sequences for comparison canbe conducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity methods of Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444 (1988) and Altschul et al. Nucleic Acids Res. 25(17): 3389-3402(1997), by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and BLAST in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., supra).

One example of a useful algorithm implementation is PILEUP. PILEUPcreates a multiple sequence alignment from a group of related sequencesusing progressive, pairwise alignments. It can also plot a dendrogramshowing the clustering relationships used to create the alignment.PILEUP uses a simplification of the progressive alignment method of Feng& Doolittle, J. Mol. Evol. 35:351-360 (1987). The method used is similarto the method described by Higgins & Sharp, CABIOS 5:151-153 (1989). Asa general rule, PileUp can align up to 500 sequences, with any singlesequence in the final alignment restricted to a maximum length of 7,000characters.

The multiple alignment procedure begins with the pairwise alignment ofthe two most similar sequences, producing a cluster of two alignedsequences. This cluster can then be aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences canbe aligned by a simple extension of the pairwise alignment of twoindividual sequences. A series of such pairwise alignments that includesincreasingly dissimilar sequences and clusters of sequences at eachiteration produces the final alignment.

“Variant” applies to both amino acid and nucleic acid sequences. Withrespect to particular nucleic acid sequences, conservatively modifiedvariants refers to those nucleic acids which encode identical oressentially identical amino acid sequences, or where the nucleic aciddoes not encode an amino acid sequence, to essentially identicalsequences. Because of the degeneracy of the genetic code, a large numberof functionally identical nucleic acids encode any given protein. Forinstance, the codons GCA, GCC, GCG and GCT all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations,” which are one species ofconservatively modified variations. Every nucleic acid sequence hereinwhich encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of skill will recognize that eachdegenerate codon in a nucleic acid can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide is implicit in each describedsequence.

Also included within the definition of target proteins of the presentinvention are amino acid sequence variants of wild-type target proteins.These variants fall into one or more of three classes: substitutional,insertional or deletional variants. These variants ordinarily areprepared by site specific mutagenesis of nucleotides in the DNA encodingthe target protein, using cassette or PCR mutagenesis or othertechniques well known in the art, to produce DNA encoding the variant,and thereafter expressing the DNA in recombinant cell culture. Varianttarget protein fragments having up to about 100-150 amino acid residuesmay be prepared by in vitro synthesis using established techniques.Amino acid sequence variants are characterized by the predeterminednature of the variation, a feature that sets them apart from naturallyoccurring allelic or interspecies variation of the target protein aminoacid sequence. The variants typically exhibit the same qualitativebiological activity as the naturally occurring analogue, althoughvariants can also be selected which have modified characteristics.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to about 20 amino acids,although considerably longer insertions may be tolerated. Deletionsrange from about 1 to about 20 residues, although in some cases,deletions may be much longer.

Substitutions, deletions, and insertions or any combinations thereof maybe used to arrive at a final derivative. Generally, these changes aredone on a few amino acids to minimize the alteration of the molecule.However, larger characteristics may be tolerated in certaincircumstances.

Individual substitutions, to a nucleic acid, peptide, polypeptide, orprotein sequence which alters a single amino acid or a small percentageof amino acids in the encoded sequence is a “conservatively modifiedvariant” where the alteration results in the substitution of an aminoacid with a chemically similar amino acid. Conservative substitutiontables providing functionally similar amino acids are well known in theart (Henikoff and Henikoff (Proc. Natl. Acad. Sci. USA 89; 10915-10919(1992))).

“Cytoskeletal component” denotes any molecule that is found inassociation with the cellular cytoskeleton, that plays a role inmaintaining or regulating the structural integrity of the cytoskeleton,or that mediates or regulates motile events mediated by thecytoskeleton. Includes cytoskeletal polymers (e.g., actin filaments,microtubules, intermediate filaments, myosin fragments), molecularmotors (e.g., kinesins, myosins, dyneins), cytoskeleton associatedregulatory proteins (e.g., tropomysin, alpha-actinin) and cytoskeletalassociated binding proteins (e.g., microtubules associated proteins,actin binding proteins).

“Cytoskeletal function” refers to biological roles of the cytoskeleton,including but not limited to the providing of structural organization(e.g., microvilli, mitotic spindle) and the mediation of motile eventswithin the cell (e.g., muscle contraction, mitotic chromosome movements,contractile ring formation and function, pseudopodal movement, activecell surface deformations, vesicle formation and translocation.)

A “diagnostic” as used herein is a compound, method, system, or devicethat assists in the identification and characterization of a health ordisease state. The diagnostic can be used in standard assays as is knownin the art.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

“High stringency conditions” may be identified by those that: (1) employlow ionic strength and high temperature for washing, for example 0.015 Msodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at50° C.; (2) employ during hybridization a denaturing agent such asformamide, for example, 50% (v/v) formamide with 0.1% bovine serumalbumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphatebuffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at42° C.; or (3) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS,and 10% dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC(sodium chloride/sodium citrate) and 50% formamide at 55° C., followedby a high-stringency wash consisting of 0.1×SSC containing EDTA at 55°C.

“High throughput screening” as used herein refers to an assay whichprovides for multiple candidate agents or samples to be screenedsimultaneously. As further described below, examples of such assays mayinclude the use of microtiter plates which are especially convenientbecause a large number of assays can be carried out simultaneously,using small amounts of reagents and samples.

By “host cell” is meant a cell that contains an expression vector andsupports the replication or expression of the expression vector. Hostcells may be prokaryotic cells such as E. coli, or eukaryotic cells suchas yeast, insect, amphibian, or mammalian cells such as CHO, HeLa andthe like, or plant cells. Both primary cells and cultured cell lines areincluded in this definition.

The phrase “hybridizing specifically to” refers to the binding,duplexing, or hybridizing of a molecule only to a particular nucleotidesequence under stringent conditions when that sequence is present in acomplex mixture (e.g., total cellular) DNA or RNA. Stringent conditionsare sequence-dependent and will be different in different circumstances.Longer sequences hybridize specifically at higher temperatures.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength, pH, and nucleic acid concentration) at which 50%of the probes complementary to the target sequence hybridize to thetarget sequence at equilibrium. Typically, stringent conditions will bethose in which the salt concentration is less than about 1.0 M sodiumion, typically about 0.05 to 1.0 M sodium ion concentration (or othersalts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. forshort probes (e.g., 10 to 50 nucleotides) and at least about 60° C. forlong probes (e.g., greater than 50 nucleotides). Stringent conditionsmay also be achieved with the addition of destabilizing agents such asformamide.

The terms “identical” or percent “identity”, in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence over a comparisonwindow, as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection. Preferably, thepercent identity exists over a region of the sequence that is at leastabout 25 amino acids in length, more preferably over a region that is atleast 50 amino acids in length. This definition also refers to thereverse complement of a test nucleic acid sequence, provided that thetest sequence has a designated or substantial identity to a referencesequence. Preferably, the percent identity exists over a region of thesequence that is at least about 25 nucleotides in length, morepreferably over a region that is at least 50 nucleotides in length.

When percentage of sequence identity is used in reference to proteins orpeptides, it is recognized that residue positions that are not identicaloften differ by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g,. charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. The scoring of conservative substitutions can becalculated according to, e.g., the algorithm of Meyers & Millers,Computer Applic. Biol. Sci. 4:11-17 (1988)

The terms “isolated”, “purified”, or “biologically pure” refer tomaterial that is substantially or essentially free from components whichnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylarnide gel electrophoresis or highperformance liquid chromatography. A protein that is the predominantspecies present in a preparation is substantially purified. In anisolated gene, the nucleic acid of interest is separated from openreading frames which flank the gene of interest and encode proteinsother than the protein of interest. The term “purified” denotes that anucleic acid or protein gives rise to essentially one band in anelectrophoretic gel. Particularly, it means that the nucleic acid orprotein is at least 85% pure, more preferably at least 95% pure, andmost preferably at least 99% pure.

A “label” is a composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include fluorescent proteins such as green, yellow, red or bluefluorescent proteins, radioisotopes such as ³²P, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens and proteins for which antisera ormonoclonal antibodies are available (e.g., the polypeptide of SEQ IDNO:2 can be made detectable, e.g., by incorporating a radio-label intothe peptide, and used to detect antibodies specifically reactive withthe peptide).

A “labeled nucleic acid probe or oligonucleotide” is one that is bound,either covalently, through a linker, or through ionic, van der Waals, orhydrogen bonds to a label such that the presence of the probe may bedetected by detecting the presence of the label bound to the probe.

“Moderately stringent conditions” may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e g., temperature, ionic strength and % SDS)less stringent than those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextransulfate, and 20 μg/mL denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C. The skilled artisan willrecognize how to adjust the temperature, ionic strength, etc. asnecessary to accommodate factors such as probe length and the like.

“Modulators,” “inhibitors,” and “activators of a target protein” referto modulatory molecules identified using in vitro and in vivo assays fortarget protein activity. Such assays include ATPase activity,microtubule gliding, microtubule depolymerizing activity, and bindingactivity such as microtubule binding activity or binding of nucleotideanalogs. Samples or assays that are treated with a candidate agent at atest and control concentration. The control concentration can be zero.If there is a change in target protein activity between the twoconcentrations, this change indicates the identification of a modulator.A change in activity, which can be an increase or decrease, ispreferably a change of at least 20% to 50%, more preferably by at least50% to 75%, more preferably at least 75% to 100%, and more preferably150% to 200%, and most preferably is a change of at least 2 to 10 foldcompared to a control. Additionally, a change can be indicated by achange in binding specificity or substrate.

“Molecular motor” refers to a molecule that utilizes chemical energy togenerate mechanical force. According to one embodiment, the molecularmotor drives the motile properties of the cytoskeleton.

The phrase “motor domain” refers to the domain of a target protein thatconfers membership in the kinesin superfamily of motor proteins asdetermined by alignment with the motor domain of true kinesin.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences as well asthe sequence explicitly indicated. For example, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260)2605-2608(1985); Cassol et al. 1992; Rossolini et al. Mol. Cell. Probes 8:91-98(1994)). The term nucleic acid is used interchangeably with gene, cDNA,and mRNA encoded by a gene.

“Nucleic acid probe or oligonucleotide” is defined as a nucleic acidcapable of binding to a target nucleic acid of complementary sequencethrough one or more types of chemical bonds, usually throughcomplementary base pairing, usually through hydrogen bond formation. Asused herein, a probe may include natural (i.e., A, G, C, T, or U) ormodified bases. In addition, the bases in a probe may be joined by alinkage other than a phosphodiester bond, so long as it does notinterfere with hybridization. Thus, for example, probes may be peptidenucleic acids in which the constituent bases are joined by peptide bondsrather than phosphodiester linkages. It will be understood by one ofskill in the art that probes may bind target sequences lacking completecomplementarity with the probe sequence depending upon the stringency ofthe hybridization conditions. The probes are preferably directly labeledwith isotopes, chromophores, lumiphores, chromogens, or indirectlylabeled such as with biotin to which a streptavidine complex may laterbind. By assaying for the presence or absence of the probe, one candetect the presence or absence of the select sequence or subsequence.

The terms “polypeptide” “peptide” and “protein” are used interchangeablyherein to refer to a polymer of amino acid residues. The terms apply toamino acid polymers in which one or more amino acid residues is anartificial chemical analogue of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers. Atarget protein comprises a polypeptide demonstrated to have at leastmicrotubule stimulated ATPase activity and, preferably that also bindsto an antibody selectively immnuoreactive with HsKif16b or whosesequence is derived from HsKif16b. by mutagenesis and/or recombination.Amino acids may be referred to herein by either their commonly known oneor three letter symbols. Nucleotides, likewise, may be referred to bytheir commonly accepted single-letter codes, i.e., the one-lettersymbols recommended by the IUPAC-IUB.

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA box element. A promoter also optionally includes distal enhancer orrepressor elements which can be located as much as several thousand basepairs from the start site of transcription. A “constitutive” promoter isa promoter that is active under most environmental and developmentalconditions. An “inducible” promoter is a promoter that is underenvironmental or developmental regulation. The term “operably linked”refers to a functional linkage between a nucleic acid expression controlsequence (such as a promoter, or array of transcription factor bindingsites) and a second nucleic acid sequence, wherein the expressioncontrol sequence directs transcription of the nucleic acid correspondingto the second sequence.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biologics. Thus, under designated immunoassay conditions, thespecified antibodies bind to a particular protein at least two times thebackground and do not substantially bind in a significant amount toother proteins present in the sample. Specific binding to an antibodyunder such conditions may require an antibody that is selected for itsspecificity for a particular protein. For example, antibodies raised toHsKif16b with the amino acid sequence encoded in SEQ ID NO:2 or SEQ IDNO:4 can be selected to obtain only those antibodies that arespecifically immunoreactive with HsKif16b and not with other proteins,except for polymorphic variants, orthologs, alleles, and closely relatedhomologues of HsKif16b. This selection may be achieved by subtractingout antibodies that cross react with molecules, for example, such as C.elegans unc-104 and human Kif1A. A variety of immunoassay formats may beused to select antibodies specifically immunoreactive with a particularprotein. For example, solid-phase ELISA immunoassays are routinely usedto select antibodies specifically immunoreactive with a protein (see,e.g., Harlow & Lane, Antibodies, A Laboratory Manual (1988), for adescription of immunoassay formats and conditions that can be used todetermine specific immunoreactivity). Typically a specific or selectivereaction will be at least twice background signal or noise and moretypically more than 10 to 100 times background.

The phrase “selectively associates with” refers to the ability of anucleic acid to “selectively hybridize” with another as defined above,or the ability of an antibody to “selectively (or specifically) bind toa protein, as defined above.

“Test composition” (used interchangeably herein with “candidate agent”and “test compound” and “test agent”) refers to a molecule orcomposition whose effect on the interaction between one or morecytoskeletal components it is desired to assay. The “test composition”can be any molecule or mixture of molecules, optionally in a carrier.

A “therapeutic” as used herein refers to a compound which is believed tobe capable of modulating the cytoskeletal system in vivo which can haveapplication in either human or animal disease. Modulation of thecytoskeletal system would be desirable in a number of conditionsincluding, but not limited to: abnormal stimulation of endothelial cells(e.g., atherosclerosis), solid and hematopoetic tumors and tumormetastasis, benign tumors, for example, hemangiomas, acoustic neuromas,neurofibromas, pyogenic granulomas, vascular malfunctions, abnormalwound healing, inflammatory and immune disorders such as rheumatoidarthritis, Bechet's disease, gout or gouty arthritis, abnormalangiogenesis accompanying: rheumatoid arthritis, psoriasis, diabeticretinopathy, and other ocular angiogenic disesase such as, maculardegeneration, corneal graft rejection, corneal overgrowth, glaucoma, andOsler Webber syndrome.

II. The Target Protein

The present invention provides for the first time a nucleic acidencoding human HsKif16b. This protein is a member of the kinesinsuperfamily of motor proteins and the Unc-104 subfamily. Morespecifically, the HsKif16b sequence of FIG. 2 or FIG. 4 sharesapproximately 50% identity with various kinesins, being closest insequence to mouse HsKif16b (see, Nakagawa et al. Proc. Natl. Acad. Sci.94:9654 (1997) and mouse or human Kif1b.

In one aspect, HsKif16b can be defined by having at least one orpreferably more than one of the following functional and structuralcharacteristics. Functionally, HsKif16b will have microtubule-stimulatedATPase activity, and microtubule motor activity that is ATP dependent.HsKif16b activity can also be described in terms of its ability to bindmicrotubules.

The novel nucleotides sequences provided herein encode HsKif16b orfragments thereof. Thus, in one aspect, the nucleic acids providedherein are defined by the novel proteins provided herein. The proteinprovided herein comprises an amino acid sequence which has one or moreof the following characteristics: greater than 70% sequence identitywith SEQ ID NO:2 or SEQ ID NO:4, preferably greater than 80%, morepreferably greater than 90%, more preferably greater than 95% or, inanother embodiment, has 98 to 100% sequence identity with SEQ ID NO:2 orSEQ ID NO:4. As described above, when describing the nucleotide in termsof SEQ ID NO:1 or SEQ ID NO:3, the sequence identity may be slightlylower due to the degeneracy in the genetic code. Also included withinthe definition of the target proteins are amino acid sequence variantsof wild-type target proteins.

Portions of the HsKif16b nucleotide sequence may be used to identifypolymorphic variants, orthologs, alleles, and homologues of HsKif16b.This identification can be made in vitro, e.g., under stringenthybridization conditions and sequencing, or by using the sequenceinformation in a computer system for comparison with other nucleotidesequences. Sequence comparison can be performed using any of thesequence comparison algorithms discussed below, with BLAST as apreferred algorithm.

As will be appreciated by those in the art, the target proteins can bemade in a variety of ways, including both synthesis de novo and byexpressing a nucleic acid encoding the protein.

Target proteins of the present invention may also be modified in a wayto form chimeric molecules comprising a fusion of a target protein witha tag polypeptide which provides an epitope to which an anti-tagantibody can selectively bind. The epitope tag is generally placed atthe amino or carboxyl terminus of the target protein. Provision of theepitope tag enables the target protein to be readily detected, as wellas readily purified by affinity purification. Various tag epitopes arewell known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 (see, Field et al. (1988) Mol. Cell. Biol.8:2159); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (see, Evans et al., (1985) Molecular and CellularBiology, 5:3610); and the Herpes Simplex virus glycoprotein D (gD) tagand its antibody (see, Paborsky et al., (1990) Protein Engineering,3:547). Other tag polypeptides include the Flag-peptide (see, Hopp etal. (1988) BioTechnology 6:1204); the KT3 epitope peptide (see, Martineet al. (1992) Science, 255:192); tubulin epitope peptide (see, Skinner(1991) J. Biol. Chem. 266:15173); and the T7 gene 10 protein peptide tag(see, Lutz-Freyermuth et al. (1990) Proc. Natl. Acad. Sci. USA 87:6393.

The biological activity of any of the peptides provided herein can beroutinely confirmed by the assays provided herein such as those whichassay ATPase activity or microtubule binding activity. In oneembodiment, polymorphic variants, alleles, and orthologs, homologues ofHsKif16b are confirmed by using a ATPase or microtubule binding assaysas known in the art.

The isolation of biologically active HsKif16b for the first timeprovides a means for assaying for modulators of this kinesin superfamilyprotein. Biologically active HsKif16b is useful for identifyingmodulators of HsKif16b or fragments thereof and kinesin superfamilymembers using in vitro assays such as microtubule gliding assays, ATPaseassays (Kodama et al., J. Biochem. 99:1465-1472 (1986); Stewart et al.,Proc. Nat'l Acad. Sci. USA 90:5209-5213 (1993)), and binding assaysincluding microtubule binding assays (Vale et al., Cell 42:39-50(1985)). In vivo assays and uses are provided herein as well. Alsoprovided herein are methods of identifying candidate agents which bindto HsKif16b and portions thereof.

As further described herein, a wide variety of assays, therapeutic anddiagnostic methods are provided herein which utilize the novel compoundsdescribed herein. The uses and methods provided herein, as furtherdescribed below have in vivo, in situ, and in vitro applications, andcan be used in medicinal, veterinary, agricultural and research basedapplications.

III. Isolation of the gene encoding HsKif16b

A. General Recombinant DNA Methods

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)), Methods in Enzymology Vol 266 (R. F.Doolittle, ed., 1996).

For nucleic acids, sizes are given in either kilobases (kb) or basepairs (bp). These are estimates derived from agarose or acrylamide gelelectrophoresis, from sequenced nucleic acids, or from published DNAsequences. For proteins, sizes are given in kilodaltons (kDa) or aminoacid residue numbers. Proteins sizes are estimated from gelelectrophoresis, from mass spectroscopy, from sequenced proteins, fromderived amino acid sequences, or from published protein sequences.

Oligonucleotides that are not commercially available can be chemicallysynthesized according to the solid phase phosphoramidite triester methodfirst described by Beaucage & Caruthers, Tetrahedron Letts. 22:1859-1862(1981), using an automated synthesizer, as described in Van Devanter etal., Nucleic Acids Res. 12:6159-6168 (1984). Purification ofoligonculeotides is by either native acrylamide gel electrophoresis orby anion-exchange HPLC as described in Pearson & Reanier, J. Chrom.225:137-149 (1983).

The sequence of the cloned genes and synthetic oligonucleotides can beverified after cloning using, e.g., the chain termination method forsequencing double-stranded templates of Wallace et al., Gene 16:21-26(1981).

B. Cloning Methods for the Isolation of Nucleotide Sequences EncodingHsKif16b

In general, the nucleic acid sequences encoding HsKif16b and relatednucleic acid sequence homologs are cloned from cDNA and genomic DNAlibraries or isolated using amplification techniques witholigonucleotide primers. Alternatively, expression libraries can be usedto clone HsKif16b and HsKif16b homologues by detected expressedhomologues immunologically with antisera or purified antibodies madeagainst HsKif16b that also recognize and selectively bind to theHsKif16b homologue. Finally, amplification techniques using primers canbe used to amplify and isolate HsKif16b from DNA or RNA. Amplificationtechniques using degenerate primers can also be used to amplify andisolate HsKif16b homologues. Amplification techniques using primers canalso be used to isolate a nucleic acid encoding HsKif16b. These primerscan be used, e.g., to amplify a probe of several hundred nucleotides,which is then used to screen a library for full-length HsKif16b.

Appropriate primers and probes for identifying the gene encodinghomologues of HsKif16b in other species are generated from comparisonsof the sequences provided herein. As described above, antibodies can beused to identify HsKif16b homologues. For example, antibodies made tothe motor domain of HsKif16b or to the whole protein are useful foridentifying HsKif16b homlogues.

To make a cDNA library, one should choose a source that is rich in themRNA of choice, e.g., HsKif16b. The mRNA is then made into cDNA usingreverse transcriptase, ligated into a recombinant vector, and introducedinto a recombinant host for propagation, screening and cloning. Methodsfor making and screening cDNA libraries are well known (see, e.g.,Gubler & Hoffman, Gene 25: 263-269); Sambrook et al., supra; Ausubel etal., supra).

For a genomic library, the DNA is extracted from the tissue and eithermechanically sheared or enzymatically digested to yield fragments ofabout 12-20 kb. The fragments are then separated by gradientcentrifugation from undesired sizes and are constructed in bacteriophagelambda vectors. These vectors and phage are packaged in vitro.Recombinant phage are analyzed by plaque hybridization as described inBenton & Davis, Science 196:180-182 (1977). Colony hybridization is readout as generally described in Grunstein et al., Proc. Natl. Acad. Sci.USA, 72:3961-3965 (1975).

An alternative method of isolating HsKif16b nucleic acid and itshomologues combines the use of synthetic oligonucleotide primers andamplification of an RNA or DNA template (see U.S. Pat. Nos. 4,683,195and 4,683,202; PCR Protocols: A guide to Methods and Applications (Inniset al., eds. 1990)). Methods such as polymerase chain reaction andligase chain reaction can be used to amplify nucleic acid sequences ofHsKif16b directly from mRNA, from cDNA, from genomic libraries or cDNAlibraries. Degenerate oligonucleotides can be designed to amplifyHsKif16b homologues using the sequences provided herein. Restrictionendonuclease sites can be incorporated into the primers. Polymerasechain reaction or other in vitro amplification methods may also beuseful, for example, to clone nucleic acid sequences that code forproteins to be expressed, to make nucleic acids to use as probes fordetecting the presence of HsKif16b encoding mRNA in physiologicalsamples, for nucleic sequencing or for other purposes. Genes amplifiedby the PCR reaction can be purified from agarose gels and cloned into anappropriate vector.

Gene expression of HsKif16b can also be analyzed by techniques known inthe art, e.g., reverse transcription and amplification of mRNA,isolation of total RNA or poly A+RNA, northern blotting, dot blotting,in situ hybridization, RNase protection, quantitative PCR, and the like.

Synthetic oligonucleotides can be used to construct recombinant HsKif16bgenes for use as probes or for expression of protein. This method isperformed using a series of overlapping oligonucleotides usually 40-120bp in length, representing both the sense and nonsense strands of thegene. These DNA fragments are then annealed, ligated and cloned.Alternatively, amplification techniques can be used with precise primersto amplify a specific subsequence of the HsKif16b gene. The specificsubsequence is then ligated into an expression vector.

The gene for HsKif16b is typically cloned into intermediate vectorsbefore transformation into prokaryotic or eukaryotic cells forreplication and/or expression. The intermediate vectors are typicallyprokaryote vectors or shuttle vectors.

C. Expression in Prokaryotes and Eukaryotes

To obtain high level expression of a cloned gene, such as those cDNAsencoding HsKif16b, it is important to construct an expression vectorthat contains a strong promoter to direct transcription, atranscription/translation terminator, and if for a nucleic acid encodinga protein, a ribosome binding site for translational initiation.Suitable bacterial promoters are well known in the art and described,e.g., in Sambrook et al. and Ausubel et al. Bacterial expression systemsfor expressing the HsKif16b protein are available in, e.g., E. coli,Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983);Mosbach et al., Nature 302:543-545 (1983). Kits for such expressionsystems are commercially available. Eukaryotic expression systems formammalian cells, yeast, and insect cells are well known in the art andare also commercially available. The pET expression system (Novagen) isa preferred prokaryotic expression system.

The promoter used to direct expression of a heterologous nucleic aciddepends on the particular application. The promoter is preferablypositioned about the same distance from the heterologous transcriptionstart site as it is from the transcription start site in its naturalsetting. As is known in the art, however, some variation in thisdistance can be accommodated without loss of promoter function.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the HsKif16b encodingnucleic acid in host cells. A typical expression cassette thus containsa promoter operably linked to the nucleic acid sequence encodingHsKif16b and signals required for efficient polyadenylation of thetranscript, ribosome binding sites, and translation termination. Thenucleic acid sequence encoding HsKif16b may typically be linked to acleavable signal peptide sequence to promote secretion of the encodedprotein by the transformed cell. Such signal peptides would include,among others, the signal peptides from tissue plasminogen activator,insulin, and neuron growth factor, and juvenile hormone esterase ofHeliothis virescens. Additional elements of the cassette may includeenhancers and, if genomic DNA is used as the structural gene, intronswith functional splice donor and acceptor sites.

In addition to a promoter sequence, the expression cassette should alsocontain a transcription termination region downstream of the structuralgene to provide for efficient termination. The termination region may beobtained from the same gene as the promoter sequence or may be obtainedfrom different genes.

The particular expression vector used to transport the geneticinformation into the cell is not particularly critical. Any of theconventional vectors used for expression in eukaryotic or prokaryoticcells may be used. Standard bacterial expression vectors includeplasmids such as pBR322 based plasmids, pSKF, pET23, and fusionexpression systems such as GST and LacZ. Epitope tags can also be addedto recombinant proteins to provide convenient methods of isolation,e.g., c-myc or histidine tags.

Expression vectors containing regulatory elements from eukaryoticviruses are typically used in eukaryotic expression vectors, e.g., SV40vectors, cytomegalovirus vectors, papilloma virus vectors, and vectorsderived from Epstein Bar virus. Other exemplary eukaryotic vectorsinclude pMSG, pAV009/A⁺, pMTO10/A⁺, pMAMneo-5, baculovirus pDSVE, andany other vector allowing expression of proteins under the direction ofthe SV40 early promoter, SV40 late promoter, CMV promoter,metallothionein promoter, murine mammary tumor virus promoter, Roussarcoma virus promoter, polyhedrin promoter, or other promoters showneffective for expression in eukaryotic cells.

Some expression systems have markers that provide gene amplificationsuch as thymidine kinase, hygromycin B phosphotransferase, anddihydrofolate reductase. Alternatively, high yield expression systemsnot involving gene amplification are also suitable, such as using abaculovirus vector in insect cells, with a HsKif16b encoding sequenceunder the direction of the polyhedrin promoter or other strongbaculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of eukaryotic sequences. The particularantibiotic resistance gene chosen is not critical, any of the manyresistance genes known in the art are suitable. The prokaryoticsequences are preferably chosen such that they do not interfere with thereplication of the DNA in eukaryotic cells, if necessary.

Standard transfection or transformation methods are used to producebacterial, mammalian, yeast or insect cell lines that express largequantities of HsKif16b protein, which are then purified using standardtechniques (see, e.g., Colley et al., J. Biol. Chem. 264:17619-17622(1989); Guide to Protein Purification, in Methods in Enzymology, vol.182 (Deutscher ed., 1990)).

Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison, J. Bact.,132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology,101:347-362 (Wu et al., eds, 1983). Any of the well known procedures forintroducing foreign nucleotide sequences into host cells may be used.These include the use of calcium phosphate transfection, polybrene,protoplast fusion, electroporation, liposomes, microinjection, plasmavectors, viral vectors and any of the other well known methods forintroducing cloned genomic DNA, cDNA, synthetic DNA or other foreigngenetic material into a host cell (see, e.g., Sambrook et al., supra).It is only necessary that the particular genetic engineering procedureused be capable of successfully introducing at least one gene into thehost cell capable of expressing HsKif16b.

After the expression vector is introduced into the cells, thetransfected cells are cultured under conditions favoring expression ofHsKif16b, which is recovered from the culture using standard techniquesidentified below.

IV. Purification of HsKif16b Protein

Either naturally occurring or recombinant HsKif16b can be purified foruse in functional assays. In a preferred embodiment, the target proteinsare purified for use in the assays to provide substantially puresamples. Alternatively, the target protein need not be substantiallypure as long as the sample comprising the target protein issubstantially free of other components that can contribute to theproduction of ADP or phosphate.

The target proteins may be isolated or purified in a variety of waysknown to those skilled in the art depending on what other components arepresent in the sample. Standard purification methods includeelectrophoretic, molecular, immunological, and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, chromatofocussing, selectiveprecipitation with such substances as ammonium sulfate;and others (see,e.g., Scopes, Protein Purification: Principles and Practice (1982); U.S.Pat. No. 4,673,641; Ausubel et al. supra; and Sambrook et al., supra).For example, the target protein can be purified using a standardanti-target antibody column. Ultrafiltration and diafiltrationtechniques, in conjunction with protein concentration, are also useful.A preferred method of purification is use of Ni-NTA agarose (Qiagen).

The expressed protein can be purified by standard chromatographicprocedures to yield a purified, biochemically active protein. Theactivity of any of the peptides provided herein can be routinelyconfirmed by the assays provided herein such as those which assay ATPaseactivity or microtubule binding activity. Biologically active targetprotein is useful for identifying modulators of target protein orfragments thereof and kinesin superfamily members using in vitro assayssuch as microtubule gliding assays, ATPase assays (Kodama et al., J.Biochem. 99:1465-1472 (1986); Stewart et al., Proc. Nat'l Acad. Sci. USA90:5209-5213 (1993)), and binding assays including microtubule bindingassays (Vale et al., Cell 42:39-50 (1985)), as described in detailbelow.

A. Purification of HsKif16b from Recombinant Bacteria

Recombinant proteins are expressed by transformed bacteria in largeamounts, typically after promoter induction; but expression can beconstitutive. Promoter induction with IPTG is a preferred method ofexpression. Bacteria are grown according to standard procedures in theart. Fresh or frozen bacteria cells are used for isolation of protein.Alternatively, it is possible to purify HsKif16b from bacteriaperiplasm. After HsKif16b is exported into the periplasm of thebacteria, the periplasmic fraction of the bacteria can be isolated bycold osmotic shock in addition to other methods known to skill in theart. To isolate recombinant proteins from the periplasm, the bacterialcells are centrifuged to form a pellet. The pellet is resuspended in abuffer containing 20% sucrose. To lyse the cells, the bacteria arecentrifuged and the pellet is resuspended in ice-cold 5 mM MgSO₄ andkept in an ice bath for approximately 10 minutes. The cell suspension iscentrifuged and the supernatant decanted and saved. The recombinantproteins present in the supernatant can be separated from the hostproteins by standard separation techniques well known to those of skillin the art.

Suitable purification schemes for some specific kinesins are outlined inU.S. Ser. No. 09/295,612, filed Apr. 20, 1999, hereby expresslyincorporated herein in its entirety for all purposes.

B. Standard Protein Separation Techniques For Purifying HsKif16b

Solubility Fractionation

Often as an initial step, particularly if the protein mixture iscomplex, an initial salt fractionation can separate many of the unwantedhost cell proteins (or proteins derived from the cell culture media)from the recombinant protein of interest. The preferred salt is ammoniumsulfate. Ammonium sulfate precipitates proteins by effectively reducingthe amount of water in the protein mixture. Proteins then precipitate onthe basis of their solubility. The more hydrophobic a protein is, themore likely it is to precipitate at lower ammonium sulfateconcentrations. A typical protocol includes adding saturated ammoniumsulfate to a protein solution so that the resultant ammonium sulfateconcentration is between 20-30%. This concentration will precipitate themost hydrophobic of proteins. The precipitate is then discarded (unlessthe protein of interest is hydrophobic) and ammonium sulfate is added tothe supernatant to a concentration known to precipitate the protein ofinterest. The precipitate is then solubilized in buffer and the excesssalt removed if necessary, either through dialysis or diafiltration.Other methods that rely on solubility of proteins, such as cold ethanolprecipitation, are well known to those of skill in the art and can beused to fractionate complex protein mixtures.

Size Differential Filtration

The molecular weight of HsKif16b can be used to isolated it fromproteins of greater and lesser size using ultrafiltration throughmembranes of different pore size (for example, Amicon or Milliporemembranes). As a first step, the protein mixture is ultrafilteredthrough a membrane with a pore size that has a lower molecular weightcut-off than the molecular weight of the protein of interest. Theretentate of the ultrafiltration is then ultrafiltered against amembrane with a molecular cut off greater than the molecular weight ofthe protein of interest. The recombinant protein will pass through themembrane into the filtrate. The filtrate can then be chromatographed asdescribed below.

Column Chromatography

HsKif16b can also be separated from other proteins on the basis of itssize, net surface charge, hydrophobicity, and affinity for ligands. Inaddition, antibodies raised against proteins can be conjugated to columnmatrices and the proteins immunopurified. All of these methods are wellknown in the art. It will be apparent to one of skill thatchromatographic techniques can be performed at any scale and usingequipment from many different manufacturers (e.g., Pharmacia Biotech).

V. Immunological Detection of HsKif16b

In addition to the detection of HsKif16b genes and gene expression usingnucleic acid hybridization technology, one can also use immunoassays todetect HsKif16b. Immunoassays can be used to qualitatively orquantitatively analyze HsKif16b. A general overview of the applicabletechnology can be found in Harlow & Lane, Antibodies: A LaboratoryManual (1988).

A. Antibodies to HsKif16b

Methods of producing polyclonal and monoclonal antibodies that reactspecifically with HsKif16b are known to those of skill in the art (see,e.g., Coligan, Current Protocols in Immunology (1991); Harlow & Lane,supra; Goding, Monoclonal Antibodies: Principles and Practice (2d ed.1986); and Kohler & Mustein, Nature 256:495-497 (1975). Such techniquesinclude antibody preparation by selection of antibodies from librariesof recombinant antibodies in phage or similar vectors, as well aspreparation of polyclonal and monoclonal antibodies by immunizingrabbits or mice (see, e.g., Huse et al., Science 246:1275-1281 (1989);Ward et al., Nature 341:544-546 (1989)).

A number of HsKif16b comprising immunogens may be used to produceantibodies specifically reactive with HsKif16b. For example, recombinantHsKif16b or a antigenic fragment thereof such as the motor domain, isisolated as described herein. Recombinant protein can be expressed ineukaryotic or prokaryotic cells as described above, and purified asgenerally described above. Recombinant protein is the preferredimmunogen for the production of monoclonal or polyclonal antibodies.Alternatively, a synthetic peptide derived from the sequences disclosedherein and conjugated to a carrier protein can be used an immunogen.Naturally occurring protein may also be used either in pure or impureform. The product is then injected into an animal capable of producingantibodies. Either monoclonal or polyclonal antibodies may be generated,for subsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those ofskill in the art. An inbred strain of mice (e.g., BALB/C mice) orrabbits is immunized with the protein using a standard adjuvant, such asFreund's adjuvant, and a standard immunization protocol. The animal'simmune response to the immunogen preparation is monitored by taking testbleeds and determining the titer of reactivity to HsKif16b. Whenappropriately high titers of antibody to the immunogen are obtained,blood is collected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to theprotein can be done if desired (see Harlow & Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell (see Kohler & Milstein, Eur. J. Immunol. 6:511-519 (1976)).Alternative methods of immortalization include transformation withEpstein Barr Virus, oncogenes, or retroviruses, or other methods wellknown in the art. Colonies arising from single immortalized cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, one may isolate DNA sequences which encode a monoclonalantibody or a binding fragment thereof by screening a DNA library fromhuman B cells according to the general protocol outlined by Huse et al.,Science 246:1275-1281 (1989).

Monoclonal antibodies and polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Typically, polyclonal antisera with a titer of 10⁴ or greater areselected and tested for their cross reactivity against non-HsKif16bproteins or even other homologous proteins from other organisms (e.g.,C. elegans unc-104 or human Kif1A), using a competitive bindingimmunoassay. Specific polyclonal antisera and monoclonal antibodies willusually bind with a K_(d) of at least about 0.1 mM, more usually atleast about 1 μM, preferably at least about 0.1 μM or better, and mostpreferably, 0.01 μM or better.

Once HsKif16b specific antibodies are available, HsKif16b can bedetected by a variety of immunoassay methods. For a review ofimmunological and immunoassay procedures, see Basic and ClinicalImmunology (Stites & Terr eds., 7th ed. 1991). Moreover, theimmunoassays of the present invention can be performed in any of severalconfigurations, which are reviewed extensively in Enzyme Immunoassay(Maggio ed., 1980); and Harlow & Lane, supra.

B. Binding Assays

Antibodies can be used for treatment or to identify the presence ofHsKif16b having the sequence identity characteristics as describedherein. Additionally, antibodies can be used to identify modulators ofthe interaction between the antibody and HsKif16b as further describedbelow. While the following discussion is directed toward the use ofantibodies in the use of binding assays, it is understood that the samegeneral assay formats such as those described for “non-competitive” or“competitive” assays can be used with any compound which binds toHsKif16b such as microtubules or the compounds described in Ser. No.60/070,772.

In a preferred embodiment, HsKif16b is detected and/or quantified usingany of a number of well recognized immunological binding assays (see,e.g., U.S. Pat. Nos. 4,366,241; 4,376,110; 4,517,288; and 4,837,168).For a review of the general immunoassays, see also Methods in CellBiology Volume 37. Antibodies in Cell Biology (Asai, ed. 1993); Basicand Clinical Immunology (Stites & Terr, eds., 7th ed. 1991).Immunological binding assays (or immunoassays) typically use an antibodythat specifically binds to a protein or antigen of choice (in this casethe HsKif16b or antigenic subsequence thereof). The antibody (e.g.,anti-HsKif16b) may be produced by any of a number of means well known tothose of skill in the art and as described above.

Immunoassays also often use a labeling agent to specifically bind to andlabel the complex formed by the antibody and antigen. The labeling agentmay itself be one of the moieties comprising the antibody/antigencomplex. Thus, the labeling agent may be a labeled HsKif16b polypeptideor a labeled anti-HsKif16b antibody. Alternatively, the labeling agentmay be a third moiety, such a secondary antibody, that specificallybinds to the antibody/HsKif16b complex (a secondary antibody istypically specific to antibodies of the species from which the firstantibody is derived). Other proteins capable of specifically bindingimmunoglobulin constant regions, such as protein A or protein G may alsobe used as the label agent. These proteins exhibit a strongnon-immunogenic reactivity with immunoglobulin constant regions from avariety of species (see generally Kronval et al., J. Immunol. 111:1401-1406 (1973); Akerstrom et al., J. Immunol. 135:2589-2542 (1985)).The labeling agent can be modified with a detectable moiety, such asbiotin, to which another molecule can specifically bind, such asstreptavidin. A variety of detectable moieties are well known to thoseskilled in the art.

Throughout the assays, incubation and/or washing steps may be requiredafter each combination of reagents. Incubation steps can vary from about5 seconds to several hours, preferably from about 5 minutes to about 24hours. However, the incubation time will depend upon the assay format,antigen, volume of solution, concentrations, and the like. Usually, theassays will be carried out at ambient temperature, although they can beconducted over a range of temperatures, such as 4° C. to 40° C.

Non-Competitive Assay Formats

Immunoassays for detecting HsKif16b in samples may be either competitiveor noncompetitive. Noncompetitive immunoassays are assays in which theamount of antigen is directly measured. In one preferred “sandwich”assay, for example, the anti-HsKif16b antibodies can be bound directlyto a solid substrate on which they are immobilized. These immobilizedantibodies then capture HsKif16b present in the test sample. HsKif16b isthus immobilized is then bound by a labeling agent, such as a secondHsKif16b antibody bearing a label. Alternatively, the second antibodymay lack a label, but it may, in turn, be bound by a labeled thirdantibody specific to antibodies of the species from which the secondantibody is derived. The second or third antibody is typically modifiedwith a detectable moiety, such as biotin, to which another moleculespecifically binds, e.g., streptavidin, to provide a detectable moiety.

Competitive assay formats

In competitive assays, the amount of HsKif16b present in the sample ismeasured indirectly by measuring the amount of a known, added(exogenous) HsKif16b displaced (competed away) from an anti-HsKif16bantibody by the unknown HsKif16b present in a sample. In one competitiveassay, a known amount of HsKif16b is added to a sample and the sample isthen contacted with an antibody that specifically binds to HsKif16b. Theamount of exogenous HsKif16b bound to the antibody is inverselyproportional to the concentration of HsKif16b present in the sample. Ina particularly preferred embodiment, the antibody is immobilized on asolid substrate. The amount of HsKif16b bound to the antibody may bedetermined either by measuring the amount of HsKif16b present in aHsKif16b/antibody complex, or alternatively by measuring the amount ofremaining uncomplexed protein. The amount of HsKif16b may be detected byproviding a labeled HsKif16b molecule.

A hapten inhibition assay is another preferred competitive assay. Inthis assay the known HsKif16b, is immobilized on a solid substrate. Aknown amount of anti-HsKif16b antibody is added to the sample, and thesample is then contacted with the HsKif16b. The amount of anti-HsKif16bantibody bound to the known immobilized HsKif16b is inverselyproportional to the amount of HsKif16b present in the sample. Again, theamount of immobilized antibody may be detected by detecting either theimmobilized fraction of antibody or the fraction of the antibody thatremains in solution. Detection may be direct where the antibody islabeled or indirect by the subsequent addition of a labeled moiety thatspecifically binds to the antibody as described above.

Cross-reactivity determinations

Immunoassays in the competitive binding format can also be used forcrossreactivity determinations. For example, a protein at leastpartially encoded by SEQ ID NO:2 can be immobilized to a solid support.Proteins (e.g., C. elegans unc-104 or human Kif1A) are added to theassay that compete for binding of the antisera to the immobilizedantigen. The ability of the added proteins to compete for binding of theantisera to the immobilized protein is compared to the ability ofHsKif16b encoded by SEQ ID NO:2 to compete with itself. The percentcrossreactivity for the above proteins is calculated, using standardcalculations. Those antisera with less than 10% crossreactivity witheach of the added proteins listed above are selected and pooled. Thecross-reacting antibodies are optionally removed from the pooledantisera by immunoabsorption with the added considered proteins, e.g.,distantly related homologues.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps the protein of this invention, to the immunogenprotein (i.e., HsKif16b of SEQ ID NO:2). In order to make thiscomparison, the two proteins are each assayed at a wide range ofconcentrations and the amount of each protein required to inhibit 50% ofthe binding of the antisera to the immobilized protein is determined. Ifthe amount of the second protein required to inhibit 50% of binding isless than 10 times the amount of the protein encoded by SEQ ID NO:2 thatis required to inhibit 50% of binding, then the second protein is saidto specifically bind to the polyclonal antibodies generated to aHsKif16b immunogen.

Other Assay Formats

Western blot (immunoblot) analysis is used to detect and quantify thepresence of HsKif16b in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind HsKif16b. The anti-HsKif16b antibodiesspecifically bind to the HsKif16b on the solid support. These antibodiesmay be directly labeled or alternatively may be subsequently detectedusing labeled antibodies (e.g., labeled sheep anti-mouse antibodies)that specifically bind to the anti-HsKif16b antibodies.

Other assay formats include liposome immunoassays (LIA), which useliposomes designed to bind specific molecules (e.g., antibodies) andrelease encapsulated reagents or markers. The released chemicals arethen detected according to standard techniques (see Monroe et al., Amer.Clin. Prod. Rev. 5:34-41 (1986)).

Reduction of Non-specific Binding

One of skill in the art will appreciate that it is often desirable tominimize non-specific binding in immunoassays. Particularly, where theassay involves an antigen or antibody immobilized on a solid substrateit is desirable to minimize the amount of non-specific binding to thesubstrate. Means of reducing such non-specific binding are well known tothose of skill in the art. Typically, this technique involves coatingthe substrate with a proteinaceous composition. In particular, proteincompositions such as bovine serum albumin (BSA), nonfat powdered milk,and gelatin are widely used with powdered milk being most preferred.

Labels

The particular label or detectable group used in the assay is not acritical aspect of the invention, as long as it does not significantlyinterfere with the specific binding of the antibody used in the assay.The detectable group can be any material having a detectable physical orchemical property. Such detectable labels have been well-developed inthe field of immunoassays and, in general, most any label useful in suchmethods can be applied to the present invention. Thus, a label is anycomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Useful labels inthe present invention include magnetic beads (e.g., DYNABEADS™),fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red,rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, or³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase andothers commonly used in an ELISA), calorimetric labels such as colloidalgold or colored glass or plastic beads (e.g., polystyrene,polypropylene, latex, etc.) or other labels that can be detected by massspectroscopy, NMR spectroscopy, or other analytical means known in theart.

The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. As indicatedabove, a wide variety of labels may be used, with the choice of labeldepending on sensitivity required, ease of conjugation with thecompound, stability requirements, available instrumentation, anddisposal provisions.

Non-radioactive labels are often attached by indirect means. Generally,a ligand molecule (e.g., biotin) is covalently bound to the molecule.The ligand then binds to another molecules (e.g., streptavidin)molecule, which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. The ligands and their targets can be used inany suitable combination with antibodies that recognize HsKif16b, orsecondary antibodies that recognize anti-HsKif16b.

The molecules can also be conjugated directly to signal generatingcompounds, e.g., by conjugation with an enzyme or fluorophore. Enzymesof interest as labels will primarily be hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see U.S. Pat.No. 4,391,904.

Means of detecting labels are well known to those of skill in the art.Thus, for example, where the label is a radioactive label, means fordetection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by means of photographic film, by the use ofelectronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. Finally simple calorimetriclabels may be detected simply by observing the color associated with thelabel. Thus, in various dipstick assays, conjugated gold often appearspink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. Forinstance, agglutination assays can be used to detect the presence of thetarget antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

VI. Assays for Modulators of the Target Protein

A. Functional Assays

Assays that can be used to test for modulators of the target proteininclude a variety of in vitro or in vivo assays, e.g., microtubulegliding assays, binding assays such as microtubule binding assays,microtubule depolymerization assays, and ATPase assays (Kodama et al.,J. Biochem. 99: 1465-1472 (1986); Stewart et al., Proc. Nat'l Acad. Sci.USA 90: 5209-5213 (1993); (Lombillo et al., J. Cell Biol. 128:107-115(1995); (Vale et al., Cell 42:39-50 (1985)).

Modulation is tested by screening for candidate agents capable ofmodulating the activity of the target protein comprising the steps ofcombining a candidate agent with the target protein, as above, anddetermining an alteration in the biological activity of the targetprotein. Thus, in this embodiment, the candidate agent should both bindto the target protein (although this may not be necessary), and alterits biological or biochemical activity as defined herein. The methodsinclude both in vitro screening methods and in vivo screening of cellsfor alterations in cell cycle distribution, cell viability, or for thepresence, morphology, activity, distribution, or amount of mitoticspindles, as are generally outlined above.

In a preferred embodiment, molecular motor activity is measured by themethods disclosed in Ser. No. 09/314,464, filed May 18, 1999, entitled“Compositions and assay utilizing ADP or phosphate for detecting proteinmodulators”, which is incorporated herein by reference in its entirety.More specifically, this assay detects modulators of any aspect of akinesin motor function ranging from interaction with microtubules tohydrolysis of ATP. ADP or phosphate is used as the readout for proteinactivity.

There are a number of enzymatic assays known in the art which use ADP asa substrate. For example, kinase reactions such as pyruvate kinases areknown. See, Nature 78:632 (1956) and Mol. Pharmacol. 6:31 (1970). Thisis a preferred method in that it allows the regeneration of ATP. In oneembodiment, the level of activity of the enzymatic reaction isdetermined directly. In a preferred embodiment, the level of activity ofthe enzymatic reaction which uses ADP as a substrate is measuredindirectly by being coupled to another reaction. For example, in oneembodiment, the method further comprises a lactate dehydrogenasereaction under conditions which normally allow the oxidation of NADH,wherein said lactate dehydrogenase reaction is dependent on the pyruvatekinase reaction. Measurement of enzymatic reactions by coupling is knownin the art. Furthermore, there are a number of reactions which utilizephosphate. Examples of such reactions include a purine nucleosidephosphorylase reaction. This reaction can be measured directly orindirectly. A particularly preferred embodiments utilizes the pyruvatekinase/lactate dehydrogenase system.

In one embodiment, the detection of the ADP or phosphate proceedsnon-enzymatically, for example, by binding or reacting the ADP orphosphate with a detectable compound. For example, phosphomolybdatebased assays may be used which involve conversion of free phosphate to aphosphomolybdate complex. One method of quantifying the phosphomolybdateis with malachite green. Alternatively, a fluorescently labeled form ofa phosphate binding protein, such as the E. coli phosphate bindingprotein, can be used to measure phosphate by a shift in itsfluorescence.

In addition, target protein activity can be examined by determiningmodulation of target protein in vitro using cultured cells. The cellsare treated with a candidate agent and the effect of such agent on thecells is then determined either directly or by examining relevantsurrogate markers. For example, characteristics such as mitotic spindlemorphology and cell cycle distribution can be used to determine theeffect.

Thus, in a preferred embodiment, the methods comprise combining a targetprotein and a candidate agent, and determining the effect of thecandidate agent on the target protein. Generally a plurality of assaymixtures are run in parallel with different agent concentrations toobtain a differential response to the various concentrations. Typically,one of these concentrations serves as a negative control, i.e., at zeroconcentration or below the level of detection.

As will be appreciated by those in the art, the components may be addedin buffers and reagents to assay target protein activity and giveoptimal signals. Since the methods allow kinetic measurements, theincubation periods can be optimized to give adequate detection signalsover the background.

In a preferred embodiment, an antifoam or a surfactant is included inthe assay mixture. Suitable antifoams include, but are not limited to,antifoam 289 (Sigma). Suitable surfactants include, but are not limitedto, Tween, Tritons, including Triton X-100, saponins, andpolyoxyethylene ethers. Generally, the antifoams, detergents, orsurfactants are added at a range from about 0.01 ppm to about 10 ppm.

A preferred assay design is also provided. In one aspect, the inventionprovides a multi-time-point (kinetic) assay, with at least two datapoints being preferred. In the case of multiple measurements, theabsolute rate of the protein activity can be determined.

B. Binding Assays

In a preferred embodiment, the binding of the candidate agent isdetermined through the use of competitive binding assays. In thisembodiment, the competitor is a binding moiety known to bind to thetarget protein, such as an antibody, peptide, binding partner, ligand,etc. Under certain circumstances, there may be competitive binding asbetween the candidate agent and the binding moiety, with the bindingmoiety displacing the candidate agent.

Competitive screening assays may be done by combining the target proteinand a drug candidate in a first sample. A second sample comprises acandidate agent, the target protein and a compound that is known tomodulate the target protein. This may be performed in either thepresence or absence of microtubules. The binding of the candidate agentis determined for both samples, and a change, or difference in bindingbetween the two samples indicates the presence of an agent capable ofbinding to the target protein and potentially modulating its activity.That is, if the binding of the candidate agent is different in thesecond sample relative to the first sample, the candidate agent iscapable of binding to the target protein.

In one embodiment, the candidate agent is labeled. Either the candidateagent, or the competitor, or both, is added first to the target proteinfor a time sufficient to allow binding. Incubations may be performed atany temperature which facilitates optimal activity, typically between 4and 40° C. Incubation periods are selected for optimum activity, but mayalso be optimized to facilitate rapid high throughput screening.Typically between 0.1 and 1 hour will be sufficient. Excess reagent isgenerally removed or washed away. The second component is then added,and the presence or absence of the labeled component is followed, toindicate binding.

In a preferred embodiment, the competitor is added first, followed bythe candidate agent. Displacement of the competitor is an indication thecandidate agent is binding to the target protein and thus is capable ofbinding to, and potentially modulating, the activity of the targetprotein. In this embodiment, either component can be labeled. Thus, forexample, if the competitor is labeled, the presence of label in the washsolution indicates displacement by the agent. Alternatively, if thecandidate agent is labeled, the presence of the label on the supportindicates displacement.

In an alternative embodiment, the candidate agent is added first, withincubation and washing, followed by the competitor. The absence ofbinding by the competitor may indicate the candidate agent is bound tothe target protein with a higher affinity. Thus, if the candidate agentis labeled, the presence of the label on the support, coupled with alack of competitor binding, may indicate the candidate agent is capableof binding to the target protein.

C. Candidate Agents

Candidate agents encompass numerous chemical classes, though typicallythey are organic molecules, preferably small organic compounds having amolecular weight of more than 100 and less than about 2,500 daltons.Candidate agents comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group,preferably at least two of the functional chemical groups. The candidateagents often comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof. Particularly preferred are peptides.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. In a preferred embodiment,the candidate agents are organic chemical moieties, a wide variety ofwhich are available in the literature.

D. Other Assay Components

The assays provided utilize target protein as defined herein. In oneembodiment, portions of target protein are utilized; in a preferredembodiment, portions having target protein activity as described hereinare used. In addition, the assays described herein may utilize eitherisolated target proteins or cells or animal models comprising the targetproteins.

A variety of other reagents may be included in the screening assays.These include reagents like salts, neutral proteins, e.g. albumin,detergents, etc which may be used to facilitate optimal protein-proteinbinding and/or reduce non-specific or background interactions. Also,reagents that otherwise improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.,may be used. The mixture of components may be added in any order thatprovides for the requisite binding.

VII. Applications

The methods of the invention are used to identify compounds useful inthe treatment of cellular proliferation diseases. Disease states whichcan be treated by the methods and compositions provided herein include,but are not limited to, cancer (further discussed below), autoimmunedisease, arthritis, graft rejection, inflammatory bowel disease,proliferation induced after medical procedures, including, but notlimited to, surgery, angioplasty, and the like. It is appreciated thatin some cases the cells may not be in a hyper or hypo proliferationstate (abnormal state) and still require treatment. For example, duringwound healing, the cells may be proliferating “normally”, butproliferation enhancement may be desired. Similarly, as discussed above,in the agriculture arena, cells may be in a “normal” state, butproliferation modulation may be desired to enhance a crop by directlyenhancing growth of a crop, or by inhibiting the growth of a plant ororganism which adversely affects the crop. Thus, in one embodiment, theinvention herein includes application to cells or individuals afflictedor impending affliction with any one of these disorders or states.

The compositions and methods provided herein are particularly deemeduseful for the treatment of cancer including solid tumors such as skin,breast, brain, cervical carcinomas, testicular carcinomas, etc. Moreparticularly, cancers that may be treated by the compositions andmethods of the invention include, but are not limited to: Cardiac:sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma),myxoma, rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogeniccarcinoma (squamous cell, undifferentiated small cell, undifferentiatedlarge cell, adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchialadenoma, sarcoma, lymphoma, chondromatous hamartoma, mesotheliorna;Gastrointestinal: esophagus (squamous cell carcinoma, adenocarcinoma,leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel(adenocarcinoma, lymphoma, carcinoid tumors, Karposi's sarcoma,leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), large bowel(adenocarcinoma, tubular adenoma, villous adenoma, hamartoma,leiomyoma); Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinorna,hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma; Bone:osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma [serouscystadenocarcinorna, mucinous cystadenocarcinoma, unclassifiedcarcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma], fallopian tubes (carcinoma); Hematologic:blood (myeloid leukemia [acute and chronic], acute lymphoblasticleukemia, chronic lymphocytic leukemia, myeloproliferative diseases,multiple myeloma, myelodysplastic syndrome), Hodgkin's disease,non-Hodgkin's lymphoma [malignant lymphoma]; Skin: malignant melanoma,basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, molesdysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis;and Adrenal glands: neuroblastoma. Thus, the term cancerous cell” asprovided herein, includes a cell afflicted by any one of the aboveidentified conditions.

Accordingly, the compositions of the invention are administered tocells. By “administered” herein is meant administration of atherapeutically effective dose of the candidate agents of the inventionto a cell either in cell culture or in a patient. By “therapeuticallyeffective dose” herein is meant a dose that produces the effects forwhich it is administered. The exact dose will depend on the purpose ofthe treatment, and will be ascertainable by one skilled in the art usingknown techniques. As is known in the art, adjustments for systemicversus localized delivery, age, body weight, general health, sex, diet,time of administration, drug interaction and the severity of thecondition nay be necessary, and will be ascertainable with routineexperimentation by those skilled in the art. By “cells” herein is meantalmost any cell in which mitosis or meiosis can be altered.

A “patient” for the purposes of the present invention includes bothhumans and other animals, particularly mammals, and other organisms.Thus the methods are applicable to both human therapy and veterinaryapplications. In the preferred embodiment the patient is a mammal, andin the most preferred embodiment the patient is human.

Candidate agents having the desired pharmacological activity may beadministered in a physiologically acceptable carrier to a patient, asdescribed herein. Depending upon the manner of introduction, thecompounds may be formulated in a variety of ways as discussed below. Theconcentration of therapeutically active compound in the formulation mayvary from about 0.1-100 wt. %. The agents maybe administered alone or incombination with other treatments, i.e., radiation, or otherchemotherapeutic agents.

In a preferred embodiment, the pharmaceutical compositions are in awater soluble form, such as pharmaceutically acceptable salts, which ismeant to include both acid and base addition salts.

The pharmaceutical compositions can be prepared in various forms, suchas granules, tablets, pills, suppositories, capsules, suspensions,salves, lotions and the like. Pharmaceutical grade organic or inorganiccarriers and/or diluents suitable for oral and topical use can be usedto make up compositions containing the therapeutically-active compounds.Diluents known to the art include aqueous media, vegetable and animaloils and fats. Stabilizing agents, wetting and emulsifying agents, saltsfor varying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

The administration of the candidate agents of the present invention canbe done in a variety of ways as discussed above, including, but notlimited to, orally, subcutaneously, intravenously, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, or intraocularly. In some instances, for example,in the treatment of wounds and inflammation, the candidate agents may bedirectly applied as a solution or spray.

One of skill in the art will readily appreciate that the methodsdescribed herein also can be used for diagnostic applications. Adiagnostic as used herein is a compound or method that assists in theidentification and characterization of a health or disease state inhumans or other animals.

The present invention also provides for kits for screening formodulators of the target protein. Such kits can be prepared from readilyavailable materials and reagents. For example, such kits can compriseany one or more of the following materials: biologically active targetprotein, reaction tubes, and instructions for testing activity of thetarget protein. Preferably, the kit contains biologically active targetprotein. A wide variety of kits and components can be prepared accordingto the present invention, depending upon the intended user of the kitand the particular needs of the user. For example, the kit can betailored for ATPase assays, microtubule gliding assays, or microtubulebinding assays.

VIII. Examples

This assay is based on detection of ADP production from a targetprotein's microtubule stimulated ATPase. ATP production is monitored bya coupled enzyme system consisting of pyruvate kinase and lactatedehydrogenase. Under the assay conditions described below, pyruvatekianse catalyzes the conversion of ADP and phosphoenol pyruvate topyruvate and ATP. Lactate dehydrogenase then catalyzes theoxidation-reduction reaction of pyruvate and NADH to lactate and NAD+.Thus, for each molecule of ADP produced, one molecule of NADH isconsumed. The amount of NADH in the assay solution is monitored bymeasuring light absorbance at a wavelength of 340 nm.

The final 25 μl assay solution consists of the following: 5 μg/ml targetprotein, 30 μg/ml microtubules, 5 μM Taxol, 0.8 mM NADH, 1.5 mMphosphoenol pyruvate, 3.5 U/ml pyruvate kinase, 5 U/ml lactatedehydrogenase, 25 mM Pipes/KOH pH 6.8, 2 mM MgCl₂, 1 mM EGTA, 1 mM MDTT,0.1 mg/ml BSA, 0.001% antifoam 289, and 1 mM ATP.

Potential candidate agents are dissolved in DMSO at a concentration ofabout 1 mg/ml and 0.5 μl of each chemical solution is dispensed into asingle well of a clear 384 well plate. Each of the 384 wells are thenfilled with 20 μl of a solution consisting of all of the assaycomponents described above except for ATP. The plate is agitated at ahigh frequency. To start the assay, 5 μl of a solution containing ATP isadded to each well. The plate is agitated and the absorbance is read at340 nm over various time intervals. The assay is run at roomtemperature.

The assay components and the performance of the assay are optimizedtogether to match the overall read time with the rate of the targetprotein's ADP production. The read time should be long enough for therate of NADH consumption to reach steady state beyond an initial lagtime of several seconds.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety.

4 1 4176 DNA Human 1 atggcatcgg tcaaggtggc cgtgagggtc cggcccatgaatcgcaggga aaaggacttg 60 gaggccaagt tcattattca gatggagaaa agcaaaacgacaatcacaaa cttaaagata 120 ccagaaggag gcactgggga ctcaggaaga gaacggaccaagaccttcac ctatgacttt 180 tctttttatt ctgctgatac aaaaagccca gattacgtttcacaagaaat ggttttcaaa 240 accctcggca cagatgtcgt gaagtctgca tttgaaggttataatgcttg tgtctttgca 300 tatgggcaaa ctggatctgg aaagtcatac actatgatgggaaattctgg agattctggc 360 ttaatacctc ggatctgtga aggactcttc agtcggataaatgaaaccac cagatgggat 420 gaagcttctt ttcgaactga agtcagctac ttagaaatttataacgaacg tgtgagagat 480 ctacttcggc ggaagtcatc taaaaccttc aatttgagagtccgtgagca tcccaaagaa 540 ggcccttatg ttgaggattt atccaaacat ttagtacagaattatggtga cgtagaagaa 600 cttatggatg cgggcaatat caaccggacc accgcagcgactgggatgaa cgacgtcagt 660 agcaggtctc atgccatctt caccatcaag ttcactcaggctaaatttga ttctgaaatg 720 ccatgtgaaa ccgtcagtaa gatccacttg gttgatcttgccggaagtga gcgtgcagat 780 gccaccggag ccaccggggt taggctaaag gaagggggaaatattaacaa gtcccttgtg 840 actctgggga acgtcatttc tgccttagct gatttatctcaggatgctgc aaatactctt 900 gcaaagaaga agcaagtttt cgtgccttac agggattctgtgttgacttg gttgttaaaa 960 gatagccttg gaggaaactc taaaactatc atgattgccaccatttcacc tgctgatgtc 1020 aattatggag aaaccctaag tactcttcgc tatgcaaatagagccaaaaa catcatcaac 1080 aagcctacca ttaatgagga tgccaacgtc aaacttatccgtgagctgcg agctgaaata 1140 gccagactga aaacgctgct tgctcaaggg aatcagattgccctcttaga ctcccccaca 1200 gctttaagta tggaggaaaa acttcagcag aatgaagcaagagttcaaga attgaccaag 1260 gaatggacaa ataagtggaa tgaaacccaa aatattttgaaagaacaaac tctagccctc 1320 aggaaagaag ggattggagt tgttttggat tctgaactgcctcatttgat tggcatcgat 1380 gatgaccttt tgagtactgg aatcatctta tatcatttaaaggaaggtca gacatacgtt 1440 ggtagagacg atgcttccac ggagcaagat attgttcttcatggccttga cttggagagt 1500 gagcattgca tctttgaaaa tatcgggggg acagtgactctgatacccct gagtgggtcc 1560 cagtgctctg tgaatggtgt tcagatcgtg gaggccacacatctaaatca aggtgctgtg 1620 attctcttgg gaagaaccaa tatgtttcgc tttaaccatccaaaggaagc cgccaagctc 1680 agggagaaga ggaagagtgg ccttctgtcc tccttcagcttgtccatgac cgacctctcg 1740 aagtcccgtg agaacctgtc tgcagtcatg ttgtataaccccggacttga atttgagagg 1800 caacagcgtg aagaacttga aaaattagaa agtaaaaggaaactcataga agaaatggag 1860 gaaaagcaga aatcagacaa ggctgaactg gagcggatgcagcaggaggt ggagacccag 1920 cgcaaggaga cagaaatcgt gcagctccag attcgcaagcaggaggagag cctcaaacgc 1980 cgcagcttcc acatcgagaa caagctaaag gatttacttgcggagaagga aaaatttgaa 2040 gaggagaggc tgagggaaca gcaggaaatc gagctgcagaagaagagaca agaagaagag 2100 acctttctcc gcgtccaaga agaactccaa cgactcaaagaactcaacaa caacgagaag 2160 gctgagaagt ttcagatatt tcaagaactg gaccagctccaaaaggaaaa agatgaacag 2220 tatgccaagc ttgaactgga aaaaaagaga ctagaggagcaggagaagga gcaggtcatg 2280 ctcgtggccc atctggaaga gcagctccga gagaagcaggagatgatcca gctcctgcgg 2340 cgtggggagg tacagtgggt ggaagaggag aagagggacctggaaggcat tcgggaatcc 2400 ctcctgcggg tgaaggaggc tcgtgccgga ggggatgaagatggcgagga gttagaaaag 2460 gctcaactgc gtttcttcga attcaagaga aggcagcttgtcaagctagt gaacttggag 2520 aaggacctgg ttcagcagaa agacatcctg aaaaaagaagtccaagaaga acaggagatc 2580 ctagagtgtt taaaatgtga acatgacaaa gaatctagattgttggaaaa acatgatgag 2640 agtgtcacag atgtcacgga agtgcctcaa gatttcgagaaaataaagcc agtggagtac 2700 aggctgcaat ataaagaacg ccagctacag tacctcctgcagaatcactt gccaactctg 2760 ttggaagaaa agcagagagc atttgaaatt cttgacagaggccctctcag cttagacaac 2820 actctttatc aagtagaaaa ggaaatggaa gaaaaagaagaacagcttgc acagtaccag 2880 gccaatgcaa accagctgca aaagctccaa gccacctttgaattcactgc caacattgca 2940 cgtcaggagg aaaaagtgag gaaaaaggaa aaggagattttggagtccag agagaagcag 3000 cagagagagg cgctggagcg ggccctggcc aggctggagaggagacattc tgcgctgcag 3060 aggcactcca ccctgggcac ggagattgaa gagcagaggcagaaacttgc cagtctgaac 3120 agtggcagca gagagcagtc agggctccag gctagcctggaggctgagca ggaagccctg 3180 gagaaggacc aggagaggtt agaatatgaa atccagcagctgaaacagaa gatttatgag 3240 gtcgatggtg ttcaaaaaga tcatcatggg accctggaagggaaggtggc ttcttccagc 3300 ttgccagtca gtgctgaaaa atcacacctg gttcccctcatggatgccag gatcaatgct 3360 tacattgaag aagaagtcca aagacgcctt caggatttgcatcgtgtgat tagtgaaggc 3420 tgcagtacat ctgcagacac gatgaaggat aatgagaaacttcacaatgg caccattcaa 3480 cgtaaactaa aatatgagct gtgtcgtgac ctcctgtgtgtcctgatgcc agagcctgat 3540 gccgctgcct gcgctaatca tcccttgctc cagcaagatctggttcagct ttctcttgat 3600 tggaaaacag aaatccctga tttagttttg ccaaatggagttcaggtgtc atccaaattc 3660 cagactacct tggttgacat gatttacttt cttcatggaaatatggaagt caatgtccct 3720 tccctggcag aagttcagtt actgctctac acaacagtgaaagtcatggg tgactctggc 3780 catgaccagt gccagtcgct agtccttctg aacacccacattgcactggt gaaggaagac 3840 tgtgtttttt atccacgcat tcgatctcga aacatacctcctccgggtgc acaatttgat 3900 gtgatcaaat gccatgcttt aagtgaattc aggtgtgttgttgttccaga aaagaaaaat 3960 gtgtcaacag tagaactagt cttcttacag aaactcaaaccttcagtggg ttccagaaat 4020 agtccacctg agcaccttca ggaagcccca aatgtccagttgttcaccac cccattgtat 4080 cttcaaggca gtcagaatgt cgcacctgag gtctggaaacttactttcaa ttctcaagat 4140 gaggctcttt ggctaatctc acatttgaca agactc 41762 1375 PRT Human 2 Met Ala Ser Val Lys Val Ala Val Arg Val Arg Pro MetAsn Arg Arg 1 5 10 15 Glu Lys Asp Leu Glu Ala Lys Phe Ile Ile Gln MetGlu Lys Ser Lys 20 25 30 Thr Thr Ile Thr Asn Leu Lys Ile Pro Glu Gly GlyThr Gly Asp Ser 35 40 45 Gly Arg Glu Arg Thr Lys Thr Phe Thr Tyr Asp PheSer Phe Tyr Ser 50 55 60 Ala Asp Thr Lys Ser Pro Asp Tyr Val Ser Gln GluMet Val Phe Lys 65 70 75 80 Thr Leu Gly Thr Asp Val Val Lys Ser Ala PheGlu Gly Tyr Asn Ala 85 90 95 Cys Val Phe Ala Tyr Gly Gln Thr Gly Ser GlyLys Ser Tyr Thr Met 100 105 110 Met Gly Asn Ser Gly Asp Ser Gly Leu IlePro Arg Ile Cys Glu Gly 115 120 125 Leu Phe Ser Arg Ile Asn Glu Thr ThrArg Trp Asp Glu Ala Ser Phe 130 135 140 Arg Thr Glu Val Ser Tyr Leu GluIle Tyr Asn Glu Arg Val Arg Asp 145 150 155 160 Leu Leu Arg Arg Lys SerSer Lys Thr Phe Asn Leu Arg Val Arg Glu 165 170 175 His Pro Lys Glu GlyPro Tyr Val Glu Asp Leu Ser Lys His Leu Val 180 185 190 Gln Asn Tyr GlyAsp Val Glu Glu Leu Met Asp Ala Gly Asn Ile Asn 195 200 205 Arg Thr ThrAla Ala Thr Gly Met Asn Asp Val Ser Ser Arg Ser His 210 215 220 Ala IlePhe Thr Ile Lys Phe Thr Gln Ala Lys Phe Asp Ser Glu Met 225 230 235 240Pro Cys Glu Thr Val Ser Lys Ile His Leu Val Asp Leu Ala Gly Ser 245 250255 Glu Arg Ala Asp Ala Thr Gly Ala Thr Gly Val Arg Leu Lys Glu Gly 260265 270 Gly Asn Ile Asn Lys Ser Leu Val Thr Leu Gly Ala Lys Lys Lys Gln275 280 285 Val Phe Val Pro Tyr Arg Asp Ser Val Leu Thr Trp Leu Leu LysAsp 290 295 300 Ser Leu Gly Gly Asn Ser Lys Thr Ile Met Ile Ala Thr IleSer Pro 305 310 315 320 Ala Asp Val Asn Tyr Gly Glu Thr Leu Ser Thr LeuArg Tyr Ala Asn 325 330 335 Arg Ala Lys Asn Ile Ile Asn Lys Pro Thr IleAsn Glu Asp Ala Asn 340 345 350 Val Lys Leu Ile Arg Glu Leu Arg Ala GluIle Ala Arg Leu Lys Thr 355 360 365 Leu Leu Ala Gln Gly Asn Gln Ile AlaLeu Leu Asp Ser Pro Thr Ala 370 375 380 Leu Ser Met Glu Glu Lys Leu GlnGln Asn Glu Ala Arg Val Gln Glu 385 390 395 400 Leu Thr Lys Glu Trp ThrAsn Lys Trp Asn Glu Thr Gln Asn Ile Leu 405 410 415 Lys Glu Gln Thr LeuAla Leu Arg Lys Glu Gly Ile Gly Val Val Leu 420 425 430 Asp Ser Glu LeuPro His Leu Ile Gly Ile Asp Asp Asp Leu Leu Ser 435 440 445 Thr Gly IleIle Leu Tyr His Leu Lys Glu Gly Gln Thr Tyr Val Gly 450 455 460 Arg AspAsp Ala Ser Thr Glu Gln Asp Ile Val Leu His Gly Leu Asp 465 470 475 480Leu Glu Ser Glu His Cys Ile Phe Glu Asn Ile Gly Gly Thr Val Thr 485 490495 Leu Ile Pro Leu Ser Gly Ser Gln Cys Ser Val Asn Gly Val Gln Ile 500505 510 Val Glu Ala Thr His Leu Asn Gln Gly Ala Val Ile Leu Leu Gly Arg515 520 525 Thr Asn Met Phe Arg Phe Asn His Pro Lys Glu Ala Ala Lys LeuArg 530 535 540 Glu Lys Arg Lys Ser Gly Leu Leu Ser Ser Phe Ser Leu SerMet Thr 545 550 555 560 Asp Leu Ser Lys Ser Arg Glu Asn Leu Ser Ala ValMet Leu Tyr Asn 565 570 575 Pro Gly Leu Glu Phe Glu Arg Gln Gln Arg GluGlu Leu Glu Lys Leu 580 585 590 Glu Ser Lys Arg Lys Leu Ile Glu Glu MetGlu Glu Lys Gln Lys Ser 595 600 605 Asp Lys Ala Glu Leu Glu Arg Met GlnGln Glu Val Glu Thr Gln Arg 610 615 620 Lys Glu Thr Glu Ile Val Gln LeuGln Ile Arg Lys Gln Glu Glu Ser 625 630 635 640 Leu Lys Arg Arg Ser PheHis Ile Glu Asn Lys Leu Lys Asp Leu Leu 645 650 655 Ala Glu Lys Glu LysPhe Glu Glu Glu Arg Leu Arg Glu Gln Gln Glu 660 665 670 Ile Glu Leu GlnLys Lys Arg Gln Glu Glu Glu Thr Phe Leu Arg Val 675 680 685 Gln Glu GluLeu Gln Arg Leu Lys Glu Leu Asn Asn Asn Glu Lys Ala 690 695 700 Glu LysPhe Gln Ile Phe Gln Glu Leu Asp Gln Leu Gln Lys Glu Lys 705 710 715 720Asp Glu Gln Tyr Ala Lys Leu Glu Leu Glu Lys Lys Arg Leu Glu Glu 725 730735 Gln Glu Lys Glu Gln Val Met Leu Val Ala His Leu Glu Glu Gln Leu 740745 750 Arg Glu Lys Gln Glu Met Ile Gln Leu Leu Arg Arg Gly Glu Val Gln755 760 765 Trp Val Glu Glu Glu Lys Arg Asp Leu Glu Gly Ile Arg Glu SerLeu 770 775 780 Leu Arg Val Lys Glu Ala Arg Ala Gly Gly Asp Glu Asp GlyGlu Glu 785 790 795 800 Leu Glu Lys Ala Gln Leu Arg Phe Phe Glu Phe LysArg Arg Gln Leu 805 810 815 Val Lys Leu Val Asn Leu Glu Lys Asp Leu ValGln Gln Lys Asp Ile 820 825 830 Leu Lys Lys Glu Val Gln Glu Glu Gln GluIle Leu Glu Cys Leu Lys 835 840 845 Cys Glu His Asp Lys Glu Ser Arg LeuLeu Glu Lys His Asp Glu Ser 850 855 860 Val Thr Asp Val Thr Glu Val ProGln Asp Phe Glu Lys Ile Lys Pro 865 870 875 880 Val Glu Tyr Arg Leu GlnTyr Lys Glu Arg Gln Leu Gln Tyr Leu Leu 885 890 895 Gln Asn His Leu ProThr Leu Leu Glu Glu Lys Gln Arg Ala Phe Glu 900 905 910 Ile Leu Asp ArgGly Pro Leu Ser Leu Asp Asn Thr Leu Tyr Gln Val 915 920 925 Glu Lys GluMet Glu Glu Lys Glu Glu Gln Leu Ala Gln Tyr Gln Ala 930 935 940 Asn AlaAsn Gln Leu Gln Lys Leu Gln Ala Thr Phe Glu Phe Thr Ala 945 950 955 960Asn Ile Ala Arg Gln Glu Glu Lys Val Arg Lys Lys Glu Lys Glu Ile 965 970975 Leu Glu Ser Arg Glu Lys Gln Gln Arg Glu Ala Leu Glu Arg Ala Leu 980985 990 Ala Arg Leu Glu Arg Arg His Ser Ala Leu Gln Arg His Ser Thr Leu995 1000 1005 Gly Thr Glu Ile Glu Glu Gln Arg Gln Lys Leu Ala Ser LeuAsn Ser 1010 1015 1020 Gly Ser Arg Glu Gln Ser Gly Leu Gln Ala Ser LeuGlu Ala Glu Gln 1025 1030 1035 1040 Glu Ala Leu Glu Lys Asp Gln Glu ArgLeu Glu Tyr Glu Ile Gln Gln 1045 1050 1055 Leu Lys Gln Lys Ile Tyr GluVal Asp Gly Val Gln Lys Asp His His 1060 1065 1070 Gly Thr Leu Glu GlyLys Val Ala Ser Ser Ser Leu Pro Val Ser Ala 1075 1080 1085 Glu Lys SerHis Leu Val Pro Leu Met Asp Ala Arg Ile Asn Ala Tyr 1090 1095 1100 IleGlu Glu Glu Val Gln Arg Arg Leu Gln Asp Leu His Arg Val Ile 1105 11101115 1120 Ser Glu Gly Cys Ser Thr Ser Ala Asp Thr Met Lys Asp Asn GluLys 1125 1130 1135 Leu His Asn Gly Thr Ile Gln Arg Lys Leu Lys Tyr GluLeu Cys Arg 1140 1145 1150 Asp Leu Leu Cys Val Leu Met Pro Glu Pro AspAla Ala Ala Cys Ala 1155 1160 1165 Asn His Pro Leu Leu Gln Gln Asp LeuVal Gln Leu Ser Leu Asp Trp 1170 1175 1180 Lys Thr Glu Ile Pro Asp LeuVal Leu Pro Asn Gly Val Gln Val Ser 1185 1190 1195 1200 Ser Lys Phe GlnThr Thr Leu Val Asp Met Ile Tyr Phe Leu His Gly 1205 1210 1215 Asn MetGlu Val Asn Val Pro Ser Leu Ala Glu Val Gln Leu Leu Leu 1220 1225 1230Tyr Thr Thr Val Lys Val Met Gly Asp Ser Gly His Asp Gln Cys Gln 12351240 1245 Ser Leu Val Leu Leu Asn Thr His Ile Ala Leu Val Lys Glu AspCys 1250 1255 1260 Val Phe Tyr Pro Arg Ile Arg Ser Arg Asn Ile Pro ProPro Gly Ala 1265 1270 1275 1280 Gln Phe Asp Val Ile Lys Cys His Ala LeuSer Glu Phe Arg Cys Val 1285 1290 1295 Val Val Pro Glu Lys Lys Asn ValSer Thr Val Glu Leu Val Phe Leu 1300 1305 1310 Gln Lys Leu Lys Pro SerVal Gly Ser Arg Asn Ser Pro Pro Glu His 1315 1320 1325 Leu Gln Glu AlaPro Asn Val Gln Leu Phe Thr Thr Pro Leu Tyr Leu 1330 1335 1340 Gln GlySer Gln Asn Val Ala Pro Glu Val Trp Lys Leu Thr Phe Asn 1345 1350 13551360 Ser Gln Asp Glu Ala Leu Trp Leu Ile Ser His Leu Thr Arg Leu 13651370 1375 3 1077 DNA Human 3 atggcatcgg tcaaggtggc cgtgagggtc cggcccatgaatcgcaggga aaaggacttg 60 gaggccaagt tcattattca gatggagaaa agcaaaacgacaatcacaaa cttaaagata 120 ccagaaggag gcactgggga ctcaggaaga gaacggaccaagaccttcac ctatgacttt 180 tctttttatt ctgctgatac aaaaagccca gattacgtttcacaagaaat ggttttcaaa 240 accctcggca cagatgtcgt gaagtctgca tttgaaggttataatgcttg tgtctttgca 300 tatgggcaaa ctggatctgg aaagtcatac actatgatgggaaattctgg agattctggc 360 ttaatacctc ggatctgtga aggactcttc agtcggataaatgaaaccac cagatgggat 420 gaagcttctt ttcgaactga agtcagctac ttagaaatttataacgaacg tgtgagagat 480 ctacttcggc ggaagtcatc taaaaccttc aatttgagagtccgtgagca tcccaaagaa 540 ggcccttatg ttgaggattt atccaaacat ttagtacagaattatggtga cgtagaagaa 600 cttatggatg cgggcaatat caaccggacc accgcagcgactgggatgaa cgacgtcagt 660 agcaggtctc atgccatctt caccatcaag ttcactcaggctaaatttga ttctgaaatg 720 ccatgtgaaa ccgtcagtaa gatccacttg gttgatcttgccggaagtga gcgtgcagat 780 gccaccggag ccaccggggt taggctaaag gaagggggaaatattaacaa gtcccttgtg 840 actctgggga acgtcatttc tgccttagct gatttatctcaggatgctgc aaatactctt 900 gcaaagaaga agcaagtttt cgtgccttac agggattctgtgttgacttg gttgttaaaa 960 gatagccttg gaggaaactc taaaactatc atgattgccaccatttcacc tgctgatgtc 1020 aattatggag aaaccctaag tactcttcgc tatgcaaatagagccaaaaa catcatc 1077 4 359 PRT Human 4 Met Ala Ser Val Lys Val AlaVal Arg Val Arg Pro Met Asn Arg Arg 1 5 10 15 Glu Lys Asp Leu Glu AlaLys Phe Ile Ile Gln Met Glu Lys Ser Lys 20 25 30 Thr Thr Ile Thr Asn LeuLys Ile Pro Glu Gly Gly Thr Gly Asp Ser 35 40 45 Gly Arg Glu Arg Thr LysThr Phe Thr Tyr Asp Phe Ser Phe Tyr Ser 50 55 60 Ala Asp Thr Lys Ser ProAsp Tyr Val Ser Gln Glu Met Val Phe Lys 65 70 75 80 Thr Leu Gly Thr AspVal Val Lys Ser Ala Phe Glu Gly Tyr Asn Ala 85 90 95 Cys Val Phe Ala TyrGly Gln Thr Gly Ser Gly Lys Ser Tyr Thr Met 100 105 110 Met Gly Asn SerGly Asp Ser Gly Leu Ile Pro Arg Ile Cys Glu Gly 115 120 125 Leu Phe SerArg Ile Asn Glu Thr Thr Arg Trp Asp Glu Ala Ser Phe 130 135 140 Arg ThrGlu Val Ser Tyr Leu Glu Ile Tyr Asn Glu Arg Val Arg Asp 145 150 155 160Leu Leu Arg Arg Lys Ser Ser Lys Thr Phe Asn Leu Arg Val Arg Glu 165 170175 His Pro Lys Glu Gly Pro Tyr Val Glu Asp Leu Ser Lys His Leu Val 180185 190 Gln Asn Tyr Gly Asp Val Glu Glu Leu Met Asp Ala Gly Asn Ile Asn195 200 205 Arg Thr Thr Ala Ala Thr Gly Met Asn Asp Val Ser Ser Arg SerHis 210 215 220 Ala Ile Phe Thr Ile Lys Phe Thr Gln Ala Lys Phe Asp SerGlu Met 225 230 235 240 Pro Cys Glu Thr Val Ser Lys Ile His Leu Val AspLeu Ala Gly Ser 245 250 255 Glu Arg Ala Asp Ala Thr Gly Ala Thr Gly ValArg Leu Lys Glu Gly 260 265 270 Gly Asn Ile Asn Lys Ser Leu Val Thr LeuGly Asn Val Ile Ser Ala 275 280 285 Leu Ala Asp Leu Ser Gln Asp Ala AlaAsn Thr Leu Ala Lys Lys Lys 290 295 300 Gln Val Phe Val Pro Tyr Arg AspSer Val Leu Thr Trp Leu Leu Lys 305 310 315 320 Asp Ser Leu Gly Gly AsnSer Lys Thr Ile Met Ile Ala Thr Ile Ser 325 330 335 Pro Ala Asp Val AsnTyr Gly Glu Thr Leu Ser Thr Leu Arg Tyr Ala 340 345 350 Asn Arg Ala LysAsn Ile Ile 355

What is claimed is:
 1. A method for screening for modulators of a targetprotein, wherein the target protein has microtubule stimulated ATPaseactivity and comprises a sequence that has greater than 95% amino acidsequence identity to SEQ ID NO:2 or SEQ ID NO:4 as measured using asequence comparison algorithm, the method comprising the steps of:contacting the target protein with a candidate agent at a firstconcentration and determining a level of activity of the target protein;and contacting the target protein with a candidate agent at a secondconcentration and determining a level of activity of the target protein;wherein the activity is selected from the group consisting of bindingactivity or ATPase activity, and wherein a difference between the levelof activity of the target protein contacted with the first concentrationof the candidate agent and the level of activity of the target proteincontacted with the second concentration of the candidate agent indicatesthat the candidate agent modulates the activity of the target protein.2. The method of claim 1, wherein the screening occurs in a multi-wellplate as part of a high-throughput screen.
 3. The method of claim 1,wherein the target protein comprises an amino acid sequence of SEQ IDNO:2.
 4. The method of claim 1, wherein the target protein comprises anamino acid sequence of SEQ ID NO:4.
 5. The method of claim 1, whereinthe target protein has greater than 98% amino acid sequence identity toSEQ ID NO:2 as measured using a sequence comparison algorithm.
 6. Themethod of claim 1, wherein the target protein has greater than 98% aminoacid sequence identity to SEQ ID NO:4 as measured using a sequencecomparison algorithm.
 7. The method of claim 1, wherein the targetprotein has been isolated from an endogenous source.
 8. The method ofclaim 1, wherein the target protein has been produced recombinantly. 9.The method of claim 1, wherein said first concentration or said secondconcentration of the candidate agent is zero or a level below detection.10. The method of claim 1, wherein the candidate agent is an agonist.11. The method of claim 1, wherein the candidate agent is an antagonist.12. The method of claim 1, wherein the candidate agent binds to thetarget protein.
 13. The method of claim 1, wherein the target protein iscontacted with the candidate agent in vivo.
 14. The method of claim 1,wherein the target protein is contacted with the candidate agent invitro.
 15. The method of claim 1, wherein contacting the target proteinwith a candidate agent comprises adding the candidate agent to a mixturecomprising the target protein under conditions which normally allow theproduction of ADP or phosphate.
 16. The method of claim 15, whereindetermining the level of activity of the target protein comprises thesteps of: i) subjecting the mixture to an enzymatic reaction, whereinsaid enzymatic reaction uses ADP or phosphate as a substrate underconditions which normally allow the ADP or phosphate to be utilized; andii) measuring NADH consumption as a measure of ADP production, wherein achange in measure of NADH consumption between the first and secondconcentrations of the candidate agent indicates that the candidate agentis a modulator of the target protein.
 17. The method of claim 1, whereinthe candidate agent is labeled.